Magnetic protein a beads

ChIP was performed as described previously based on the protocol from the Millipore Magna ChIP kit (Malvaez et al., 2011 (link); Rogge et al., 2013 (link)). Tissue was cross-linked with 1% formaldehyde (Sigma), lysed and sonicated, and chromatin was immunoprecipitated overnight with 2 μL of H3K9me3 (ab8898) or 5 μL of anti-mouse IgG (negative control, Millipore). The immunoprecipitate was collected using magnetic protein A beads (Millipore). After washing, chromatin was eluted from the beads and reverse cross-linked in the presence of proteinase K before column purification of DNA. BDNF, GABBR1, and GABRA2 promoter enrichment in ChIP samples was measured by quantitative real-time PCR using the Roche 480 LightCycler and SYBR green. Primer sequences for BDNF promoters were taken from Intlekofer et al. (2013) (link) and GABA receptor primers were designed by the Primer-BLAST program (Supplementary Table 1). Five microliters of input, anti-H3K9me3, or anti-mouse IgG immunoprecipitate from mice in each condition were examined in duplicate. To normalize ChIP-qPCR data, we used the percent input method. The input sample was adjusted to 100% and both the IP and IgG samples were calculated as a percent of this input using the formula: 100 × AE^[adjusted input –Ct (IP)]. An in-plate standard curve determined amplification efficiency (AE).

ChIP was performed as described previously (Kwapis et al., 2017 (link)) based on the protocol from the Millipore ChIP kit. Tissue was cross-linked with 1% formaldehyde (Sigma), lysed and sonicated, and chromatin was immunoprecipitated overnight with 5 μl of anti- HDAC3 (Millipore), anti-H4K8AC (Millipore) or 5 μl of anti-mouse IgG (negative control, Millipore). The immunoprecipitate was collected using magnetic protein A beads (Millipore). After washing, chromatin was eluted from the beads and reverse cross-linked in the presence of proteinase K before column purification of DNA. Fos, Nr4a1 and Nr4a2 promoter enrichment in ChIP samples was measured by quantitative real-time PCR using the Roche 480 LightCycler and SYBR green. Primer sequences for the promoters, designed by the Primer three program are listed below; 5 μl of input, anti-HDAC3 IgG, or anti-mouse IgG immunoprecipitate were examined in duplicate. To normalize ChIP-qPCR data, we used the percent input method. The input sample was adjusted to 100% and both the IP and IgG samples were calculated as a percent of this input using the formula: 100*AE^(adjusted input – Ct (IP)). An in-plate standard curve determined amplification efficiency (AE; see Table 1).

The EZ-Magna ChIP kit (EMD Millipore) was used to conduct the chromatin immunoprecipitation (ChIP) assays in accordance with the manufacturer’s protocol. After carrying out the above-mentioned cell treatments, the cells were lysed with Cell Lysis Buffer and Nuclear Lysis Buffer and sonicated to obtain chromatin fragments. Next, the lysates were immunoprecipitated with Magnetic Protein A Beads conjugated with Histone3 (Millipore) or IgG (Millipore) as a control. Western blot assay was performed to detect the enrichment of LSD1, H3K27me1 (Millipore), H3K4me2 (Millipore), and DNMT1 (Millipore) in the precipitated protein complex.

The EZ-Magna ChIP kit (EMD Millipore) was used to conduct the ChIP assays in accordance with manufacturer’s protocol, the Fadu and Tu686 cells were fixed with 4% paraformaldehyde and incubated with glycine for 10 min to generate DNA–protein cross-links. Then, the cells were lysed with Cell Lysis Buffer and Nuclear Lysis Buffer and sonicated to generate chromatin fragments. Next, the lysates were immunoprecipitated with Magnetic Protein A Beads conjugated with H3K27me2 (Millipore), or H3K36me2-specific antibodies (Millipore), or IgG as a control. Finally, the precipitated DNA was analyzed by qRT-PCR.